Fine mapping and application of dna markers linked to a gall midge resistance gene for marker-aided selection in rice

ABSTRACT

The present invention relates to fine mapping and potential application of dna markers linked to a gall midge resistance gene gm7 for marker-aided selection in rice. Towards this, the present invention discloses a combination of novel sequence characterized amplified region (SCAR) primers for use in assay with the DNA of Rice plants in question. A cross between the gall midge resistant parent, RP2333 carrying the Gm7 gene and susceptible parent Shyamala, is developed and a F 5  progeny is raised. A polymorphic band is identified from the F 5  progeny, using AFLP that cosegregates with the susceptible phenotype. This band is eluted from the gel and cloned. The cloned AFLP fragment is sequenced and primers are developed for selectively amplifying DNA of susceptible phenotypes, thus differentiating them from the resistant phenotypes. This Gm7 gene linked marker is mapped onto chromosome 4 of rice and is also shown to be linked to Gm2 gene and the blight resistance gene, Xal through fine mapping using Yeast Artificial Chromosomes (YACS) and cosmids. This marker is present in a single copy in the susceptible parent, Shyamala. Primers developed from this marker are able to differentiate between the resistant and susceptible phenotypes in different crosses carrying different gall midge resistance genes. A number of screenings of resistant and susceptible varieties of rice with these primers show consistent polymorphism between them. The use of primers for PCR amplification of DNAs from F 3  progenies derived from crosses between three different parental lines and the primers also differentiates the resistant phenotypes from the susceptible one. The primers of the present invention therefore have a great use in marker assisted selection as they show polymorphism between resistant and susceptible plants and therefore between plants with or without gall midge resistance genes.

FIELD OF INVENTION

The present invention relates to DNA markers linked to a gall midgeresistance gene for marker-aided selection in rice. More particularly,the present invention relates to fine mapping and potential applicationof DNA markers linked to a gall midge resistance gene, Gm7, formarker-aided selection in rice. The present invention also relates tonovel primers for use in preparing the aforesaid DNA markers. Thepresent invention also relates to a method of screening rice varietiesfor susceptibility and/or resistance to gall midge.

BACKGROUND OF INVENTION

Rice is the most important crop in the world with over 1.5 billionhectares under paddy cultivation and a worldwide production of over 596million tons (FAO 1999). Rice crop in the field is subject to attack bya number of insect pests, pathogens, weeds and other harmful organisms.Several studies have reported that major yield losses of rice are oftencaused by insects alone. Of these insects, a dipteran pest, the ricegall midge (Orseolia oryzae) alone is reported to cause a damage of morethan US $ 700 million annually.

Breeding of new rice varieties resistant to gall midge is a traditionaland effective method of controlling the damage. However, one of themajor problems faced by rice breeders is the emergence of new gall midgebiotypes. Different biotypes of gall midge are morphologicallyindistinguishable and capable of interbreeding but are known to differin their reaction to genetically defined rice varieties.

Different gall midge resistance genes that provide resistance againstdifferent sets of biotypes of the gall midge have been identified anddocumented (Bentur J. S. and Amudhan S., (1996) ‘Reaction ofdifferentials of different populations of Asian rice gall midge(Orseolia oryzae) under green house condition’, Indian J Agri Sci,66:197-199). At least six non-allelic gall midge resistance genes inrice (Chaudhary B. P., Shrivastava P. S., Shrivastava M. N., Khush G.S., (1986), ‘Inheritance of resistance to gall midge in some cultivarsof rice’, Rice Genetics: Proceedings of the International Rice GeneticsSymposium, International Rice Research Institute, The Phillipines,523-528.) and 5 different biotypes of the gall midge have beenidentified and reported in India (Behura S. K., Sahu S. C., Rajamani S.,Devi A., Mago R, Nair S., and Mohan M, (1999), ‘Differentiation of Asianrice gall midge Orseolia oryzae (Wood-Mason) biotypes by sequencecharacterised amplified regions (SCARs)’, Insect Mol Biol, 8: 391-397;Sardesai N., Rajyashri K. R., Behura S. K, Nair S., aand Mohan M,(2001), ‘Genetic, physiological and molecular interactions of rice withits major dipteran pest, gall midge’, Plant Cell Tissue Org Cult, 64:115-131.). Different biotypes of gall midge are distributed in differentregions of the country and normally two or more biotypes do not occurtogether at the same geographical location. Consequently, the selectionof rice plants resistant to more than one biotype of gall midge becomesvery time consuming since screening is based on the natural occurrenceof the pest that is limited to one particular time of the year i.e.,just 2-4 months following monsoon. This makes the process of breedingand pyramiding of gall midge resistance genes labor intensive and timeconsuming. Therefore, there is an urgent need for development ofmolecular markers that are tightly linked to the gene of interest thatwould enable one to follow the gene in a cross intended to breed newresistant varieties any time of the year without depending on the annualoccurrence of insects (Mohan M., Nair S., Bhagwat A., Krishna T. G.,Yano M, Bhatia C. R, and Sasaki T., (1997a), ‘Genome mapping, molecularmarkers and marker assisted selection in crop plants’ Mol Breed, 3:87-103.).

DESCRIPTION OF PRIOR ART

The first gall midge resistance gene to be mapped using restrictionfragment length polymorphism (RFLP) and random amplified polymorphic DNA(RAPD) markers was the Gm2 gene (Mohan M., Nair S., Bentur J. S.,Prasada Rao U., Bennet J., (1994), ‘RFLP and RAPD mapping of the riceGm2 gene that confers resistance to biotype 1 of gall midge (Orseoliaoryzae)’, Theor Appl Genet, 87: 782-788.) that confers resistance tobiotypes 1 and 2 of gall midge (Bentur J. S.; and Amudhan S. (1996),Reaction of differentials of different populations of Asian rice gallmidge (Orseolia oryzae) under Green House conditions: Indian J Agric Sci66:197-199). The present inventors have also tagged and mapped the gallmidge resistance gene Gm4t that confers resistance to biotypes 1, 2 and4 (Nair et al. 1996; Mohan et al. 1997). The potential use of DNAmarkers linked to the gall midge resistance genes in marker-aidedselection (MAS) has also been demonstrated (Nair et al. 1995, 1996).

Biotyping of gall midge is traditionally achieved by observing theinfectivity pattern on a set of rice differentials or varieties. It isnot possible to morphologically differentiate the different biotypes ofgall midge and thus biotyping has been solely based on differentialinfestation patterns on specific rice hosts. This has the effect ofslowing down the process of biotype identification and consequently, theselection for rice plants resistant to more than one biotype of gallmidge, particularly since the natural occurrence of gall midge isrestricted to a 2 to 4 month period every year. This also slows down theprocess of breeding new gall midge resistant rice varieties.

OBJECTS OF THE INVENTION

Accordingly, it is an object of the present invention to provide analternative to the labour-intensive and time consuming screeningprocedures of the prior art.

It is another object of the present invention to provide a nondestructive method for screening rice populations of interest to selectvarieties, which are resistant to gall midge attack.

It is another object of the present invention to provide a method foridentification of suitable molecular markers closely linked to the gallmidge resistance genes to enable easy following of the gene in a crossintended to breed new resistant varieties any time of the year withoutdepending on the annual occurrence of the insects.

It is an important object of the present invention to provide an AFLPmarker linked to a gall midge resistance gene for marker-aided selectionin rice.

It is still another important object of the present invention to providea method for fine mapping and potential application of AFLP markerslinked to a gall midge resistance gene, Gm7, for marker-aided selectionin rice.

It is yet another object of the present invention to provide novelprimers for developing AFLP marker linked to a gall midge resistancegene for marker-aided selection in rice.

SUMMARY OF THE INVENTION

The above and other objects of the present invention are achieved by thetagging of the gene with molecular markers that are closely linked withit, which cosegregate with the desired phenotype. The present inventionprovides a combination of novel sequence characterized amplified region(SCAR) primers for use in assay with the DNA of the plants in question.A cross between the gall midge resistant parent, RP2333 carrying the Gm7gene and susceptible parent Shyamala, is developed and a F₅ progeny israised. A polymorphic band is identified from the F₅ progeny, using AFLPthat cosegregates with the susceptible phenotype. This band is elutedfrom the gel and cloned. The cloned AFLP fragment is sequenced andprimers are developed for selectively amplifying DNA of susceptiblephenotypes, thus differentiating them from the resistant phenotypes.This Gm7 gene linked marker is mapped onto chromosome 4 of rice and isalso shown to be linked to Gm2 gene and the blight resistance gene, Xal,through fine mapping using Yeast Artificial Chromosomes (YACS) andcosmids. This marker is present in a single copy in the susceptibleparent, Shyamala. Primers developed from this marker are able todifferentiate between the resistant and susceptible phenotypes indifferent crosses carrying different gall midge resistance genes. Anumber of screenings of resistant and susceptible varieties of rice withthese primers show consistent polymorphism between them. The use ofprimers for PCR amplification of DNAs from F₃ progenies derived fromcrosses between three different parental lines and the primers alsodifferentiates the resistant phenotypes from the susceptible one. Theprimers of the present invention therefore have a great use in markerassisted selection as they show polymorphism between resistant andsusceptible plants and therefore between plants with or without gallmidge resistance genes.

Thus, according to the present invention, there is provided acombination of sequence characterized amplified region (SCAR) primersfor use in marker assisted selection of rice varieties which areresistant to attack by gall midge, said primers having the sequenceshown in: Seq ID # 2 and 3 respectively: 5′ - GATCATTGGAGCAACATTCTG - 3′Seq ID # 2 and 5′ - CATTTCTAATTCTTTCTTCAA - 3′ Seq ID # 3

The present invention also provides a method for preparing combinationof sequence characterized amplified region (SCAR) primers for use inmarker assisted selection of rice varieties which are resistant toattack by gall midge which comprises subjecting genomic DNA extractedfrom rice varieties resistant to gall midge biotypes and rice varietiessusceptible to gall midge biotypes to amplified fragment lengthpolymorphism (AFLP) reactions in the presence of primers, separating theamplified product, extracting DNA therefrom and subjecting it topolymerase chain reaction in the presence of the same primers employedfor the AFLP reactions to obtain gall midge susceptible specific AFLPfragment, cloning said AFLP fragment into pGEMT vector to produce acloned AFLP insert, sequencing said cloned insert and producing saidSCAR primers from said clone employing the sequence information, whereinsaid clone has a nucleotide sequence having Seq. ID. # 1 and saidprimers have the sequence shown in Seq ID # 2 and 3 respectively: 5′ -GATCATTGGAGCAACATTCTG - 3′ Seq ID # 2 and 5′ - CATTTCTAATTCTTTCTTCAA -3′ Seq ID # 3

Preferably, said rice varieties resistant to gall midge biotypes andrice varieties susceptible to gall midge biotypes from which genomic DNAare extracted are F₅ progenies cross between a rice variety carrying thegene Gm7 conferring resistance to gall midge biotypes 1, 2 and 4 and arice variety susceptible to gall midge biotypes.

In another preferred embodiment, the cloned AFLP insert comprisessusceptibility specific AFLP fragment of 598 bp.

The present method also provides a method for screening a rice varietyto determine whether it is resistant or susceptible to gall midgebiotypes which comprises extracting DNA from said rice variety,subjecting said rice variety to a polymerase chain reactionamplification in the presence of a combination primers having thesequence shown in Seq ID # 2 and 3 respectively 5′ -GATCATTGGAGCAACATTCTG - 3′ and 5′ - CATTTCTAATTCTTTCTTCAA - 3′and determining if any fragment of said DNA was amplified, amplificationof a fragment indicating the presence of susceptible phenotype specificband thereby indicating that said rice variety is susceptible to Gallmidge biotypes and absence of amplification indicating that said ricevariety is resistant to Gall midge biotypes.

DETAILED DESCRIPTION

The present invention will now be described in greater detail withreference to the accompanying drawings in which:

FIG. 1 shows a AFLP fragment segregating with the susceptible phenotypesusing primer combination shown in Seq ID # 4 and Seq ID # 5respectively, i.e., P-CG (5′ GACTGCGTACATGCACG 3′) AND M-CTG (5′GATGAGTCCTGAGTAACTG 3′).

FIG. 2A discloses the complete nucleotide sequence of thesusceptible-specific AFLP marker, SA 598, identified in the presentinvention.

FIG. 2B shows the sequence of SCAR primers (forward and reverse)designed after sequencing the susceptible-specific marker SA598 andsequence of the F8 primers.

FIG. 3 shows Gel and Southern hybridization of HindIII and DraI digestedDNA of RP 2333 and Shyamala probed with SA598.

FIG. 4 depicts the PCR-based screening for gall midge-resistant andsusceptible progeny in F % population derived from a cross between RP2333 and Shyamala using the susceptible phenotype-specific SCAR primers.

FIG. 5 shows Southern hybridization of DraI digested Nipponbare and YACDNA, forming a contig encompassing an allele of Gm2 gene with SA 598

FIG. 6 discloses PCR-based screening for gall midge-resistant andsusceptible progeny in F5 population derived from a cross between RP2333and Shyamala using F8-specific primers.

FIG. 7 discloses Southern hybridization of DraI digested Nipponbare andYAC DNAs forming a contig encompaasing an allele of Gm2 gene with F8LB.

FIG. 8 shows a Graphical representation of a portion of the map ofchromosome 4 showing the position of Gm7-linked markers.

The present invention uses bulked segregant analysis (Paran andMichelmore 1993) in the identification of AFLP and RAPD markers linkedto a gall midge resistance gene, Gm7, in rice, that have a potentialapplication in marker aided selection. Gm7 has been mapped using adifferent strategy which involved hybridisation to Bacterial ArtificialChromosomes (BACs), Yeast Artificial Chromosomes (YACs) and cosmids thatencompass the Gm2 gene, in the indica rice variety Phalguna. Finemapping of Gm7 has been reported to reveal a linkage between Gm 7, Gm2and Xal.

In the present invention, the F₅ population used was derived employingmethods known in the art from a cross between the two indica ricevarieties, RP 2333 containing Gm 7 (resistant to gall midge biotypes 1,2 and 4; Kumar et al. 1999) and Shyamla (susceptible to all gall midgebiotypes). The scoring for resistance and susceptiblity was done underfield conditions at the Indira Gandhi Agricultural University, Raipur,India. The plants were screened for the presence and absence of gall.Plants without any galls were scored as resistant and those having evenone gall per plant were scored as susceptible. The scored plants weresubsequently harvested for DNA extraction.

RP 2333, carrying the Gm7, gene, was crossed with varieties carryingother gall midge resistance genes to study the allelic relationshipbetween Gm7 and the other gall midge resistance genes. The segregationdata of F1, F2 and F3 progenies was recorded.

DNA was isolated from field-grown plants (10 weeks old) using methodsknown in the art. An equal quantity of DNA from 12 resistant and 12susceptible F5 individuals was pooled to form the resistant andsusceptible bulks, respectively. The concentration of DNA of the twobulks and the two parental DNAs was adjusted to 10 ng/ul. Thereafter,DNA was subjected to Random Amplification.

The amplification conditions employed are well known in the art and willnot pose any problem to a skilled artisan. The reaction volume was 25ul, and 30 ng template DNA was used per reaction. All reactions werecarried out on a Perkin-Elmer Cetus DNA Thermal Cycler. Taq DNApolymerase was from Stratagene (La Jolla, Calif.). The RAPD primers usedwere from the Operon 10-mer Kits (Open Technologies, Alameda, Calif.)520 random primers of Kits A to Z were utilised in the study. The RAPDproduct were separated on a 1.1% agarose gel in 1×TBE buffer with 7.5 ulof the 25 ul reaction being loaded on the gel. Gel and buffer containedethidium bromide at a concentration of 0.5 ug/ml.

The product obtained above was subjected to AFLP reactions (AmplifiedFragment Length Polymorphism) by methods known in the art. The methoddescribed by (Vos P., Hogers R., Bleeker M, Reijans M., van de Lee T.,Hones M, Friyers A., Pot J., Peleman J., Kuiper M., and Zabeau M.,(1995), ‘AFLP: a new technique for DNA fingerprinting’, Nucleic AcidsRes, 23: 4407-4414) was particularly preferable. Briefly, genomic DNAs(500 ng) from RP2333, Shyamala, the resistant and susceptible bulks andthe progenies were digested in a reaction volume of 25 μl. The digestedand adapter ligated DNA was amplified with EcoRI or PstI and MseInon-selective primer pairs in a 50 μl reaction. The amplificationprofile was 94° C. for 30 sec, 56° C. for 30 sec and 72° C. for 1 minfor 30 cycles followed by an extension at 72° C. for 5 min. Theamplified products were diluted 10-fold in TE (10 mM Tris, 0.5 pH8.0; 1mM EDTA) and used for selective amplification. EcoRI or PstI primersused in the selective amplification were radiolabeled separately bykinasing 10 ng of each primer with [□-³²P]-ATP. Selective amplificationwas carried out with 5 μl diluted preamplification product, 1 μl oflabeled EcoRI/PstI primer and 50 ng of MseI primer. The reaction volumewas 20 μl. A total of 157 primer (MseI and EcoRI/PstI combinations wereused in this study. After PCR, 20 ul of formamide dye (containing 98%formamide, 10 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol) wasadded to the reaction. The samples were heat denatured for 5 min, snapcooled on ice and loaded onto a 6% sequencing gel containing 8M urea.The gel was dried and exposed overnight to X-OMAT-AR film (Kodak) atroom temperature.

The DNA was extracted from the gel using methods known in the art. Theputatively linked AFLP fragment was first marked out on the dried gel byaligning it with the autoradiogram and cutting out the band along withthe gel and soaking the cut piece in an eppendorf containing 100 ulsterile water for 10 min (Behura et al 2000). The gel piece was thenboiled in the water for 15 min and the tube spun at 14,000 rpm for 2min. 10 ul of 3M sodium acetate and 5 ml of glycogen (10 mg/ml) wereadded to the supernatant. This tube was then spun at 14,000 rpm for 15min and the pellet washed with 85% ethanol. The pellet was dried anddissolved in 10 ul water.

The eluted AFLP fragment was re-amplified using the same primers andreaction conditions that had revealed the polymorphism and separated on0.8% agarose. The fragment showing the correct size was excised from theagarose gel and purified using a Qiagen gel extraction kit (Qiagen,Hilder, Germany) for cloning and sequencing of AFLP fragment.

The AFLP fragment considered to be putatively linked to Gm7 was firstmarked out on the dried gel by aligning it with the autoradiogram andcut out from the gel. The DNA from this gel fragment was isolated asdescribed earlier (Behura S. K, Sahu S. C., Nair S., and Mohan M.,(2000), ‘An AFLP marker that differentiates biotypes of the Asian ricegall midge (Orseolia oryzae, Wood-Mason) is sex linked and also linkedto avirulence’, Mol Gen Genet, 263:328-334.). The isolated DNA waspelleted, washed with 85% ethanol, dried and dissolved in 10 ul ofsterile dH₂O. The DNA (5 ul) was then PCR amplified using the sameprimer pairs that generated the AFLP fragment. The PCR products were runon a 0.8% agarose gel, gel-purified using a Qiagen gel extraction kit(Qiagen, Hilder, Germany) and cloned into pGEM(T) (Promega, Madison,Wis.). The clone containing the AFLP fragment was named SA598(susceptibility specific AFLP fragment of 598 bp). DNA sequence of thecloned fragment was determined by the dideoxy chain-termination methodusing the T7 Sequence Version 2.0 DNA sequencing kit (USB, Cleveland) todevelop sequence characterized amplified region (SCAR) primers from thecloned AFLP marker. This cloned AFLP insert was named as SA598(susceptibility specific AFLP fragment of 598 bp).

Southern hybridisation of AFLP fragment with parental DNA was carried bymethods known in the art. Genomic DNA (5 ug) of RP 2333 and Shyamla wasdigested with 40U each of HindIII and DraI at 37° C. overnight. Thedigested DNA was run on a 0.8% agarose gel and blotted onto a Hybondnylon membrane as described by [Williams M. N. V., Pande N., Nair S.,Mohan M., and Bennet J., (1991)), ‘Restriction fragment lengthpolymorphism analysis of polymerase chain reaction products amplifiedfrom mapped loci of rice (Oryzae sativa L.) genomic DNA’, Theor ApplGenet, 82: 489-498.]. SA 598 was excised from the plasmid by restrictionwith ApaI and NotI and the digested product separated on 0.8% agarose in1×TBE. The SA598 band was excised from the gel, purified using a Quagengel extraction kint (Qiagen, Hilder, Germany) and reamplified with theAFLP primers. The membrane was probed with SA 598 labeled with[α³²P]dCTP using the Random Primers DNA Labeling System (BethesdaResearch Laboratories, Life Technologies USA.). After hybridisation for20 h at 650C, the membrane was washed under stringent conditions (twicein 2×SSC at room temperature for 15 min each; once in 2×SSC and 0.1% SDSat 65° C. for 20 min; once in 0.25×SSC and 0.1% SDS at 65° C. for 15min; and once in 2×SSC at room temperature briefly) andautoradiographed.

Thereafter, a BAC library constructed from the high molecular weightnuclear DNA of the rice variety IR-BB21 (Wang G-L., Holsen T. E., SongW-Y., Wand H-P., and Ronald P. C., (1995), ‘Construction of a ricebacterial artificial chromosome library and identification of cloneslinked to the Xa-21 disease resistance locus’, Plant J, 7: 525-533.) wasprobed with [α³²P] dCTP labeled SA 598 as above. The filters were washedafter hybridisation at 65° C. for 20 h under stringent conditions (twicein 2×SSC at room temperature for 15 min each; once in 2×SSC and 0.1% SDSat 65° C. for 20 min; once in 0.5×SSC and 0.1% SDS at 65° C. for 20 min;and once in 2×SSC at room temperature briefly) and autoradiographed.

Blots of contiguous stretch of DraI digested YAC DNAs from japonica ricevariety, Nipponbare (Rajyashri K. R., Nair S., Ohmido N., Fukui K.,Kurata N., Sasaki T, and Mohan M., (1998), ‘Isolation and FISH mappingof yeast artificial chromosomes (YACs) encompassing an allele of the Gm2gene for gall midge resistance in rice’, Theor Appl Genet, 97:507-514)and of Cosmid DNAs digested with DraI, from indica variety, Phalguna,encompassing the Gm2 gene (data not shown) were hybridised with the SA598 labeled as above. Filters were washed under stringent conditions asabove and autoradiographed.

The forward and reverse primers internal to the 5′ and 3′ end weredesigned from the sequence of the cloned AFLP fragment (SA 598) usingthe Oligo 4.0 software (National Biosciences) and were synthesized byIntegrated DNA Technology Inc. (USA.) These primers are shown in FIG. 2.These primers were used to amplify genomic DNA from the resistant andsusceptible parents as well as resistant and susceptible individuals ofthe F5 progeny. PCR was carried out in a 50 ul reaction volumecontaining 10 mM Tris-Cl (pH 8.0) 50 mM KCl, 1.5 mM MgCl2, 0.01%gelatin, 200 uM each dNTP, 380 nM each primer, 125 ng of template DNAand 2.5 U of Taq DNA polymerase. PCR conditions were 94° C. for 1 min,45° C. for 1 min and 72° C. for 1 min, for 30 cycles.

PCR amplification of parental and bulked DNA with primers specific toXal gene and RFLP markers (RG329, RG476, RG214, F8, F10) linked to Gm2gene was also carried out. Genomic DNA of RP2333, Shyamala and theresistant and susceptible bulks was amplified with primers specific toRG329, RG476, RG214 (Yoshimura S., Umehara Y., Kurata N., Nagamura Y.,Sasaski T., Minobe Y., and Iwata N., (1996), ‘Identification of a YACclone carrying the Xa-1 allele, a bacterial blight resistance gene inrice’, Theor Appl Genet, 93:117-122.), F8, F10 (Nair et al. 1995) andprimers from the 3′ end of Xal- a bacterial blight resistance gene fromrice (Yoshimura et al. 1996; 1998). 200 ng of template DNA was taken forthe PCR reaction. PCR conditions were 94° C. for 30 s, 51° C. for 45 sand 72° C. for 1 min; for 30 cycles.

Since only the F8-specific primers (shown in Seq D # 6 and 7) (Nair etal. 1995) showed polymorphism between RP2333 and Shyamala, genomic DNAof the resistant and susceptible individuals of the F₅ population(raised from a cross between RP2333 and Shyamala) was amplified usingthe same primers. The composition of the reaction mixture and PCRconditions were as described above. The PCR products wereelectrophoresed on a 1% agarose gel in 1×TBE. The polymorphic bandamplified from the resistant parent using the F8 primers was excisedfrom the agarose gel as mentioned above, and isolated using a Qiagen gelextraction kit (Qiagen, Hilder, Germany) and called F8LB (F8 resistancelinked band).

Southern hybridization of YACs forming contig encompassing an allele ofGm2 gene with F8LB was carried as follows:

A blot containing YAC DNAs, from clones that encompass an allele of Gm2gene, digested with DraI (Rajyashri et al. 1998), was hybridized to thegel eluted F8 polymorphic band (F8LB) after labeling with [α³²P]dCTPusing the Random Primers DNA Labeling System (Bethesda ResearchLaboratories, Life Technologies, USA). Hybridisation and washingconditions were same as above.

Cross hybridisation of SA598 with F8LB was achieved by PCR carried outusing 100 ng genomic DNA of RP2333, Shyamala and 7 each of the resistantand susceptible individuals of the F₅ population with the SCAR primersand the F8 primers in two separate reactions. PCR conditions were asmentioned for the respective sets of primers. The PCR products were runon a 1% agarose gel in 1×TBE and blotted as described above. Southernhybridization of the blot of F8-primer-amplified PCR products wascarried out with SA598 labeled with [α³²P]dCTP using the Random PrimersDNA Labeling System (Bethesda Research Laboratories, Life Technologies,USA), whereas, the blot of SCAR-primer-amplified PCR product washybridized with radiolabeled F8LB. Hybridization was carried out at 65°C. for 20 h and the filters washed under stringent conditions (twice in2×SSC at room temperature for 20 min each; once in 2×SSC and 0.1% SDS at65° C. for 20 min; once in 0.1×SSC and 0.1% SDS at 65° C. for 20 min;and once in 2×SSC at room temperature briefly) and autoradiographed.

Genetic analyses of gall midge resistance in RP2333 were carried out. F₂and F₃ segregation data for resistance to biotype 1 of gall midge incrosses involving the resistant variety RP2333 and susceptible parents,Shyamala and R2270, revealed that the resistance in RP2333 is determinedby a single dominant gene (Kumar et al. 1999). Allelic crosses betweenRP2333 and varieties Samridhi, Phalguna, Abhaya and ARC5984 having thegall midge resistance genes Gm1, Gm2, Gm4 and Gm5, respectively, showeda segregation ratio of 15:1 for resistance: susceptibility in the F₂ andof 7:8:1 for resistance:segregating:susceptible progenies in F₃,indicating the independent segregation of two dominant resistance genes.Crossing between RP2333 with RP2068-18-3-5, the variety harbouring therecessive gm3 gene, showed a segregation ratio of 13:3 forresistance:susceptibility in the F₂ and 7:8:1 in the F₃ progenies (Kumaret al. 1999) indicating the independent segregation of Gm7, the dominantresistance gene and gm3, the recessive resistance gene. This shows thatthe Gm7 gene is non-allelic to the other gall midge resistance genesreported from India.

The two parental DNAs along with the resistant and susceptible bulkswere screened using 520 RAPD primers in order to identify markers linkedto Gm7. Of these, 488 primers produced amplification products while theremaining failed to amplify. 24 primers produced resistance-specificproducts and 8 produced susceptible-specific products in the parents andthe bulked DNAs. These primers were further used to screen each of the12 different individual DNAs that constituted each of the two bulkedDNAs. However, none of these primers amplified in a phenotype-specificmanner in the individual lines that constituted the bulk therebyindicating that these markers are not closely linked to Gm 7.

In order to identify additional phenotype-specific polymorphisms AFLPwas employed. Of the 157 primer combinations used in the presentinvention, one resistance-specific amplification was identified and 4showed susceptibility-linked amplification using the EcoRI/MseIcombination. Further screening of the resistant and the susceptibleindividuals of the F₅ progeny (constituting the respective bulked DNAs)using these primer combinations revealed the absence ofphenotype-specific amplifications. Using the PstI/MseI primercombinations, 20 combinations identified resistance-specific fragmentsand 5 combinations showed susceptible-specific amplification in theparents and the bulked DNAs. Analysis of the amplification pattern ofthe individuals forming the bulks using these primer combinations alsoshowed the absence of phenotype-specific amplification except for theprimers combination P-CG×M-CTG which amplified a susceptible-specificfragment in 22 of the 24 susceptible individuals As is clear from FIG.1, the first two lanes are parents, RP2333 and Shyamala, respectively,followed by the resistant (R-Pool) and susceptible (S-pool) bulks. Theremaining lanes are the resistant and susceptible progeny of the F₅population.

For cloning and Southern Hybridization, the susceptible-specific AFLPfragment (SA598) was eluted from the gel cloned into pGEMT vector andsequenced as shown in FIG. 2. FIG. 2A shows the complete nucleotidesequence of the susceptible-specific AFLP marker, SA598. Sequenceinformation was used to design 21-mer SCAR primers. The sequence of theSCAR primers are shown in FIG. 2B. Southern hybridization of HindIII andDraI digested genomic DNAs of the parents, RP2333 and Shyamala, withSA598 revealed that it is present as a single copy in the susceptibleparent. However, the probe did not show any hybridization signal withthe genomic DNA of the resistant parent. As can be seen from FIG. 3 theregions of hybridization are indicated by arrows. It can be seen thatthe probe shows hybridization signals only in Shyamala lanes. Thefigures on the left shows molecular weight in kb.

As mentioned above, SCAR primers were designed from the sequenceinformation of SA598 and these were used in a PCR assay with the DNA ofthe parents and resistant and susceptible individuals of the F₅ progenyof the population. To the applicants' knowledge such primers of thepresent invention as well as their use in nondestructive methods fordetermining whether or not a rice variety is susceptible to gall midgeis not suggested in any prior art. In the present invention, the primersamplified a 0.55 kb fragment in the susceptible individuals. However, italso amplified this fragment in some of the resistant individualsscreened as indicated in FIG. 4, which could be because of a ricevariety incorrectly scored as a resistant individual due to low insectpressure thereon. In this figure, Panels A and B represent differentindividuals of the F₅ population. Lane M represents a 1 kb DNA markerladder. Figures on the left represent molecular weight in kb In order toascertain the chromosomal location of Gm7, mapping of SA598 wasattempted. The IR-BB21 BAC library was screened with this clone.Screening identified BACs that were a subset of the clones thathybridized to YAC probes, Y5212L and F8 (data not included; thesemarkers were earlier shown to flank Gm2 [see Rajyashri et al. 1998]).Southern hybridization of the Nipponbare YAC DNAs with SA598 showed thepresence of a single copy of this marker in the japonica varietyNipponbare and the YAC clones, Y2165 and Y5212. The results of theSouthern Hybridization are shown in FIG. 5 where the figures on the leftrepresents molecular weight in kb. The insert hybridized to twooverlapping cosmids that was previously shown to encompass the Gm2 genefrom the indica variety Phalguna (data not shown).

PCR amplification of DNAs from parents and bulks with primers specificto the 3′ end of Xal and RFLP markers linked to Gm2 was carried out. AsGm7 was shown to be linked to F8, a marker previously identified to belinked to Gm2 (Mohan et al. 1994; Rajyashri et al. 1998), it wasnecessary to determine if any of the other markers linked to Gm2 arelinked to Gm7 as well. With the parental and the bulk DNA as templates,primers specific to RG329, RG476, RG214 and Xal amplified fragments ofexpected size, but the products did not reveal any amplification lengthpolymorphism between the resistant and the susceptible phenotype. Theprimer set F10 failed to amplify at all. However, the primer set F8amplified a 1.5 kb fragment that was specific to the resistantphenotype. This can be clearly seen from FIG. 6. Panels A, B and Crepresent different individuals of the F₅ population. Lane M representsa 1 kb DNA marker ladder. Arrows indicate the polymorphicresistance-phenotype-specific (F8LB) fragment amplified by F8. Figureson the left represent molecular weight in kb. PCR amplification ofresistant and susceptible individuals of the F₅ progeny with the F8 setof primers revealed that the 1.5-kb fragment amplified in all the 23resistant individuals tested except 2, and also amplified in four of thesusceptible lines.

Probing the Dra I digested YACs, encompassing an allele of Gm2, with theresistance-linked F8 fragment (F8LB) revealed the presence of 3 bands of5.5, 4.2 and 3.2 kb in Nipponbare, Y2165 and Y5212 and one band of 5.5kb in Y3487 as can be seen from FIG. 7. Again, the figures on the rightrepresent molecular weight in kb. Southern hybridizations between thePCR products generated using F8 primer with SA598 as probe and PCRproducts generated using the novel SCAR primers of the present inventionwith F8LB as probe failed to reveal any homology between the two markers(data not shown).

Since SA598, hybridizes to the BACs, YACs and cosmids encompassing theGm2 gene, it can be concluded that this marker is linked to Gm2. Also,as SA598 hybridizes to two of the cosmids to which F8 hybridizes (datanot included), it is therefore, logical to conclude that SA598 is linkedto Gm7 and is on chromosome 4 and maps along with F8 and Xal (Yoshimuraet al. 1996) and F8LB markers as is clear from FIG. 8. In FIG. 8, thedarkly shaded bar represents the position of the YAC Y2165. The numberson the left show genetic (cM) and physical distances in this region ofchromosome 4. Gm7-linked markers are on the right of F8 with the genetic(cM) and physical (kb) distances given on the extreme left, within whichthey are present. The physical and genetic distances are as givenearlier.

Genetic analyses of the gall midge resistance gene, Gm7, revealed thatit is a dominant gene that is non-allelic to the other known gall midgeresistance genes (Kumar et al. 1999). The F₅ population was raised bycrossing parents that were different viz-a-viz reaction to differentgall midge biotypes. Screening of the parents with over 520 RAPD primersfailed to reveal any polymorphism that co-segregated with either theresistance or the susceptibility trait in the individuals of the mappingpopulation. The lack of detectable polymorphisms, that were linked togall midge resistance, between the two parents could be due to the factthat both parents, RP2333 and Shyamala, are indica varieties. It wastherefore, necessary to resort to AFLP, which is known to reveal morepolymorphisms in closely related varieties.

AFLP has been used as a DNA fingerprinting technique (Vos et al. 1995)with wide usage in plant genetic studies such as for assessment ofgenetic diversity in wheat (Barrett B. A. and Kidwell K. K., (1998),‘AFLP based genetic diversity assessment among wheat cultivars from thePacific Northwest’, Corp Sci, 38: 1261-1271.), Arabidopsis (Breyne P.,Rombaut D., van Gysel A., van Montagu M, Gerats T, (1990), ‘AFLPanalysis of genetic diversity within and between Arabidopsis thalianaecotypes’, Mol Gen Genet, 261: 627-634.) and rice (Zhu J., Gale M. D.,Quarrie S., Jackson M. T., and Bryan G. J, (1998), ‘AFLP markers for thestudy of rice biodiversity’, Theor Appl Genet, 96: 602-611; Aggarwal R.K., Brar D. S., Nandi S., Huang N., and Khush G. S., (1999), ‘Phylogenicrelationships among Oryza species revealed by AFLP markers’, Theor ApplGenet, 98: 1320-1328.), for construction of high density genetic maps ofbarley (Becker J., Vos P., Kuiper M., Salamani F., and Heun M., (1995),‘Combined mapping of AFLP and RFLP markers in barley’, Mol Gen Genet,249: 65-73.), maize (Castiglioni P., Ajmone-Marsan P., van Wijk R., andMotto M., (1999), ‘AFLP markers in a molecular linkage map of maize:codominant scoring and linkage group distribution’, Theor Appl Genet,99: 425-431.) and rice (Mackill D. J., Zhang Z., Redona E. D., andColowit P. M., (1996), ‘Level of polymorphism and genetic mapping ofAFLP markers in rice’, Genome, 39:969-977; Maheswaran M., Subudhi P. K.,Nandi S., Xu J. C., Parco A., Yang D. C., and Huang N., (1997),‘Polymorphism, distribution, and segregation of AFLP markers in adoubled haploid rice population’, Theor Appl Genet, 94:39-45) and forenrichment of DNA markers near a locus of interest in potato (BallvoraA., Hesselbach J., Niewohner J., Leister D., Salamani F., and GebhardtC., (1995), ‘Marker enrichment and high resolution map of the segment ofpotato chromosome VII harboring the nematode resistance gene Grol’, MolGen Genet, 249:82-90.), Asparagus (Reamon-Büttner S. M., SchodelmaierJ., and Jung C., (1998), ‘AFLP markers tightly linked to the sex locusin Asparagus officinalis L.’, Mol Breed, 4:91-98.), tomato (Thomas C.M., Vos P., Zabeau M., Jones D. A., Norcott K. A., Chadwick B. P., andJones J. D. G., (1995), ‘Identification of amplified restrictionfragment polymorphism (AFLP) markers tightly linked to the tomato Cf-9gene for resistance to Cladosporium fulivum’, Plant J., 8:785-794.),carrot (Bradeen J. M. and Simon P. W., (1998), ‘Conversion of an AFLPfragment linked to the carrot Y₂ locus to a simple codominant PCR basedmarker form’, Theor Appl Genet, 97:960-967.) and rice (Chen D-H., delaVina M, Inukai T., Mackill D. J., Ronald P. C., and Nelson R. J.,(1999), ‘Molecular mapping of the blast resistance gene Pi44(t), in aline derived from a durably resistant rice cultivar’, Theor Appl Genet,98:1046-1053.; Xu K., Xu X., Ronald P. C., and Mackill D. J., (2000), ‘Ahigh resolution linkage map of the vicinity of the rice submergencetolerance locus Subl.’, Mol Gen Genet, 263:681-689.). Though, in thepresent study, screening with over 150 AFLP primer pairs did revealpolymorphisms between the parents, there were very few that were linkedto either the resistant or susceptible phenotype in the segregatingpopulation. However, AFLP was successful in revealing a 598 base-pairfragment (SA598) that was segregating with the susceptible phenotype inthe present study (FIG. 1).

Using the sequence information of the AFLP marker, SA598, primers weresynthesized for the sequence characterized amplified region (SCAR)approach. Earlier, RAPD markers have been successfully converted to SCARmarkers to make them more robust and reliable (Paran I. and MichelmoreR. W., (1993), ‘Development of reliable PCR based markers linked todowny mildew resistance genes in lettuce’, Theor Appl Genet, 85:985-993;Williamson V M, Ho J-Y., Wu F. F., Miller N., and Kaloshian I., (1994),‘A PCR based marker tightly linked to the nematode resistance gene Mi intomato’, Theor Appl Genet, 87:757-763; Garcia G. M., Stalker H. T.,Shroeder E., Kochert G., (1996), ‘Identification of RAPD, SCAR and RFLPmarkers tightly linked to nematode resistance genes introgressedfromArachis cardenasii into Arachis hypogaea’, Genome, 39:836-845.; Barretet al. 1998; Vidal J. R., Delavault P., Coarer M., Defontaine A.,(2000), ‘Design of grapevine (Vitis vinifera L.) cultivar specific SCARprimers for PCR fingerprinting’, Theor Appl Genet, 101:1194-1201.). Itmay be noted that absence of susceptible phenotype specific band in oneof the susceptible individuals of the progeny and the presence of theband in some of the resistant individuals of the progeny may be eitherdue to a recombination event(s) in these phenotypes or because of someescapes as a result of insufficient insect pressure in the case of theindividuals scored as resistant.

Earlier, it was difficult to map the Gm4t gene in the population derivedfrom an indica×indica cross (Abhaya×Shyamala) due to lack ofpolymorphism between them (Mohan et al. 1997b). To overcome thisdifficulty the marker linked to Gm4t was mapped to chromosome 8 inanother mapping population obtained from a japonica×indica cross(Nipponbare×Kasalath) where sufficient polymorphism did exist (Mohan etal. 1997b). Here, faced with a similar problem of lack of polymorphismbetween the parents, mapping of the Gm7 gene by using YAC, BAC andcosmid clones was attempted which have been previously mapped tochromosome 4.

In this invention, primers specific to F8 (a marker linked to Gm2 [Mohanet al. 1994]) amplified a 1.5 kb fragment (F8LB) in the resistant parentand resistant individuals arising from a cross between RP2333 andShyamala. This indicates that Gm7 is in the vicinity of Gm2. Furtherevidence of Gm7 being present on chromosome 4 comes from the results ofhybridization of the resistance specific polymorphic band of F8 (F8LB)obtained in this study to YACs that form part of the contig encompassingan allele of Gm2 gene (FIG. 7). These results are in concurrence withthe Southern hybridization data of F8 with YAC DNAs where F8 was shownto hybridize to three Dral fragments of 6.0, 4.6 and 3.3 kb inNipponbare, Y2165 and Y5212, while the 4.6 kb fragment was absent inY3487 (Rajyashri et al. 1998). Since SA598 hybridized to two cosmidclones, which also hybridize to F8, it was of interest to note thatcross hybridisation studies revealed that there was no homology betweenF8LB and SA598, thus indicating that these markers are distinct andseparate physical entities linked to Gm7. It is interesting to note thatmarkers F8, SA598 and F8LB along with the Xal gene, all map to the samecosmids (FIG. 8). Based on the above hybridization data it could beconcluded that Gm7 is linked to Gm2 and maps to chromosome 4.

There are various reports that resistance genes to different pests andpathogens such as aphids, nematodes, bacteria, fungi and viruses arelinked and located in clusters (Dickinson M. J., Jones D. A., and JonesJ. D. G., (1993), ‘Close linkage between the Cf-2/Cf-5 and Mi resistanceloci in tomato’, Mol Plant-Microbe Int, 6:341-347; Century K. S., HolubE. B., and Staskawics B. J., (1995), ‘NDR1, a locus of Arabidopsisthaliana that is required for disease resistance to both a bacteria anda fungal pathogen’, Proc Natl Acad Sci USA, 92:6597-6601; Kaloshian L.,Lange W. H., and Williamson V. M., (1995), ‘An aphid resistance locus istightly linked to the nematode resistance gene Mi in tomato’, Proc NatlAcad Sci, USA, 92:622-625; Salmeron J. M., Oldroyd E. D. G., Rommens C.M. T., Scoofield S. R., Kim H-S., Lavelle D. T., Dahlbeck D., andStaskawicz B. J., (1996), ‘Tomato Prf is a member of the leucine richrepeat class of plant disease resistance genes and lies embedded withinthe Pto kinase gene cluster’, Cell, 86:123-133; Meyers B. C., Chin D.B., Shen K. A., Sivaramakrishnan S., Lavelle D. O., Zhang Z., andMichelmore R. W., (1998), ‘The major resistance gene cluster in lettuceis highly duplicated and spans several megabases’, Plant Cell,10:1817-1832; Mian M. A. R., Wang T., Phillips D. V., Alvernaz J., andBoerma H. R., (1999), ‘Molecular mapping of the Rcs3 gene for resistanceto frogeye leaf spot in soyabean’, Crop Sci, 39:1687-1691; Parniske M.and Jones J. D., (1999), ‘Recombination between diverged clusters of thetomato Cf-9 plant disease resistance gene family’, Proc Natl Acad Sci,USA, 96:5850-5855; Brommonschenkel S. H., Frary A., Frary A., andTanksley S. D., (2000), ‘The broad spectrum tospovirus resistance geneSw-5 of tomato is a homolog of the root-knot nematode resistance geneMi’, Mol Plant-Microbe Int, 13:1130-1138; van der Voort J. R., KanyukaK., van der Vossen E., Bendahmane A., Mooijman P., Klein-Lankhorst R.,Stiekema W., Baulcombe D., and Bakker J., (1999), ‘Tight physicallinkage of the nematode resistance gene Gpa2 and the virus resistancegene Rx on a single segment introgressed from the wild species Solanumtuberosum subsp. andigena CPC1673 into cultivated potato’, MolPlant-Microbe Interact, 12:197-206; van der Vossen E., van der Voort J.N., Kanyuka K., Bendahmane A., Sandbrink H., Baulcombe D. C., Bakker J.,Stiekema W., and Klein-Lankhorst R., (2000), ‘Homologues of a singleresistance gene cluster in potato confer resistance to distinctpathogens: a virus and a nematode’, Plant J, 23:567-576;). The markersflanking gall midge resistance gene, Gm2, and the bacterial blightresistance gene, Xal, hybridize to the same YAC clone, Y2165 (Rajyashriet al. 1998) to which both, the resistant (F8LB) and the susceptible(SA598) specific markers linked to Gm7 also hybridize, indicating alinkage between Xal and Gm7 gene as well. Thus, linkage between Xal andGm2 and between Gm2 and Gm7 suggests the presence of Gm7, Gm2 and Xal asa cluster on chromosome 4, in rice.

For marker-based screening to work effectively it is desirable to haveall the genes conferring resistance to the various biotypes of gallmidge tagged. This would enable the pyramiding of resistance genesagainst various biotypes into an elite cultivar. Pyramiding of genes isan important strategy in plant breeding for the development of newvarieties with durable resistance to several biotypes of an insect pest(Mohan et al. 1997a). The resistance and susceptibility linked markerscan be used effectively in a marker aided selection programme for thepresence of the Gm7 gene against the gall midge biotypes 1, 2 and 4.With DNA markers also available for two other major gall midgeresistance genes (Nair et al. 1995, 1996) there is a potentialapplication in marker-assisted pyramiding of the genes Gm2, Gm4t and Gm7in rice, as has been reported for blast resistance in rice (Hittalmaniet al. 2000) and greenbug resistance in wheat (Porter et al. 2000).

1. A combination of sequence characterized amplified region (SCAR)primers for use in marker assisted selection of rice varieties which aresusceptible to attack by gall midge, said primers having the sequenceshown in Seq ID Nos. 2 and
 3. 2. A method for preparing combination ofsequence characterized amplified region (SCAR) primers for use in markerassisted selection of rice varieties which are resistant to attack bygall midge which comprises subjecting genomic DNA extracted from ricevarieties resistant to gall midge biotypes and rice varietiessusceptible to gall midge biotypes to amplified fragment lengthpolymorphism (AFLP), identifying a polymorphic band using AFLP thatcosegregates with the susceptible phenotype, thereby differentiating thesusceptible varities from the resistant varieties, eluting the band andcloning it on a vector to obtain a cloned AFLP insert, sequencing saidcloned insert and producing said SCAR primers from said clone employingthe sequence information, wherein said primers have the sequence shownin Seq IDs Nos. 2 and
 3. 3. A method as claimed in claim 2, wherein saidAFLP has the nucleotide sequence as shown in Seq. ID.
 1. 4. A method asclaimed in claim 2, wherein said rice varieties resistant to gall midgebiotypes and rice varieties susceptible to gall midge biotypes fromwhich genomic DNA are extracted are Fs progenies of cross between a ricevariety carrying the gene Gm7 resistant to gall midge biotypes 1,2 and 4and a rice variety susceptible to gall midge biotypes.
 5. A method asclaimed in claim 2, wherein said cloned AFLP insert comprisessusceptibility specific AFLP fragment of 598 bp.
 6. A method as claimedin claim 2, wherein said vector is a pGEMT vector.
 7. A method forpreparing combination of sequence characterized amplified region (SCAR)primers for use in marker assisted selection of rice varieties which areresistant to attack by gall midge which comprises subjecting genomic DNAextracted from rice varieties which are Fs progenies of a cross betweenrice varieties resistant to gall midge biotypes and rice varietiessusceptible to gall midge biotypes, to random amplification, extractingthe product of random amplification and subjecting it to amplifiedfragment length polymorphism (AFLP) reactions in the presence ofprimers, separating the amplified product, extracting DNA therefrom andsubjecting it to polymerase chain reaction in the presence of the sameprimers employed for the AFLP reactions to obtain gall midge susceptiblespecific AFLP fragment, cloning said AFLP fragment into pGEMT vector toproduce a cloned AFLP insert, sequencing said cloned insert andproducing said SCAR primers from said clone employing the sequenceinformation, wherein said clone has a nucleotide sequence having Seq.ID. 1 and said primers have the sequence shown in Seq IDs Nos. 2 and 3.8. A method for screening a rice variety to determine whether it isresistance or susceptible to gall midge biotypes which comprisesextracting DNA from said rice variety, subjecting said rice variety to apolymerase chain amplification reaction in the presence of a combinationprimers having the sequence shown in Seq IDs Nos 2 and 3 and determiningif any fragment of said DNA was amplified, amplification of a fragmentindicating the presence of susceptible phenotype specific band therebyindicating that said rice variety is susceptible to Gall midge biotypesand absence of amplification indicating that said rice variety isresistant to Gall midge biotypes.
 9. A method as claimed in claim 3,wherein said cloned AFLP insert comprises susceptibility specific AFLPfragment of 598 bp.
 10. A method as claimed in claim 3, wherein saidvector is a pGEMT vector.
 11. A method as claimed in claim 4, whereinsaid vector is a pGEMT vector.
 12. A method as claimed in claim 5,wherein said vector is a pGEMT vector.